Homopolymer switches mediate adaptive mutability in mismatch repair-deficient colorectal cancer.

Mismatch repair (MMR)-deficient cancer evolves through the stepwise erosion of coding homopolymers in target genes. Curiously, the MMR genes MutS homolog 6 (MSH6) and MutS homolog 3 (MSH3) also contain coding homopolymers, and these are frequent mutational targets in MMR-deficient cancers. The impact of incremental MMR mutations on MMR-deficient cancer evolution is unknown. Here we show that microsatellite instability modulates DNA repair by toggling hypermutable mononucleotide homopolymer runs in MSH6 and MSH3 through stochastic frameshift switching. Spontaneous mutation and reversion modulate subclonal mutation rate, mutation bias and HLA and neoantigen diversity. Patient-derived organoids corroborate these observations and show that MMR homopolymer sequences drift back into reading frame in the absence of immune selection, suggesting a fitness cost of elevated mutation rates. Combined experimental and simulation studies demonstrate that subclonal immune selection favors incremental MMR mutations. Overall, our data demonstrate that MMR-deficient colorectal cancers fuel intratumor heterogeneity by adapting subclonal mutation rate and diversity to immune selection.

Comprehensive single-cell genome analysis at nucleotide resolution using the PTA Analysis Toolbox.

Detection of somatic mutations in single cells has been severely hampered by technical limitations of whole-genome amplification. Novel technologies including primary template-directed amplification (PTA) significantly improved the accuracy of single-cell whole-genome sequencing (WGS) but still generate hundreds of artifacts per amplification reaction. We developed a comprehensive bioinformatic workflow, called the PTA Analysis Toolbox (PTATO), to accurately detect single base substitutions, insertions-deletions (indels), and structural variants in PTA-based WGS data. PTATO includes a machine learning approach and filtering based on recurrence to distinguish PTA artifacts from true mutations with high sensitivity (up to 90%), outperforming existing bioinformatic approaches. Using PTATO, we demonstrate that hematopoietic stem cells of patients with Fanconi anemia, which cannot be analyzed using regular WGS, have normal somatic single base substitution burdens but increased numbers of deletions. Our results show that PTATO enables studying somatic mutagenesis in the genomes of single cells with unprecedented sensitivity and accuracy.

The Dynamics of Somatic Mutagenesis During Life in Humans.

From conception to death, human cells accumulate somatic mutations in their genomes. These mutations can contribute to the development of cancer and non-malignant diseases and have also been associated with aging. Rapid technological developments in sequencing approaches in the last few years and their application to normal tissues have greatly advanced our knowledge about the accumulation of these mutations during healthy aging. Whole genome sequencing studies have revealed that there are significant differences in mutation burden and patterns across tissues, but also that the mutation rates within tissues are surprisingly constant during adult life. In contrast, recent lineage-tracing studies based on whole-genome sequencing have shown that the rate of mutation accumulation is strongly increased early in life before birth. These early mutations, which can be shared by many cells in the body, may have a large impact on development and the origin of somatic diseases. For example, cancer driver mutations can arise early in life, decades before the detection of the malignancy. Here, we review the recent insights in mutation accumulation and mutagenic processes in normal tissues. We compare mutagenesis early and later in life and discuss how mutation rates and patterns evolve during aging. Additionally, we outline the potential impact of these mutations on development, aging and disease.

Sperm DNA damage causes genomic instability in early embryonic development.

Genomic instability is common in human embryos, but the underlying causes are largely unknown. Here, we examined the consequences of sperm DNA damage on the embryonic genome by single-cell whole-genome sequencing of individual blastomeres from bovine embryos produced with sperm damaged by γ-radiation. Sperm DNA damage primarily leads to fragmentation of the paternal chromosomes followed by random distribution of the chromosomal fragments over the two sister cells in the first cell division. An unexpected secondary effect of sperm DNA damage is the induction of direct unequal cleavages, which include the poorly understood heterogoneic cell divisions. As a result, chaotic mosaicism is common in embryos derived from fertilizations with damaged sperm. The mosaic aneuploidies, uniparental disomies, and de novo structural variation induced by sperm DNA damage may compromise fertility and lead to rare congenital disorders when embryos escape developmental arrest.

Biallelic variants in POLR3GL cause endosteal hyperostosis and oligodontia.

RNA polymerase III (Pol III) is an essential 17-subunit complex responsible for the transcription of small housekeeping RNAs such as transfer RNAs and 5S ribosomal RNA. Biallelic variants in four genes (POLR3A, POLR3B, and POLR1C and POLR3K) encoding Pol III subunits have previously been found in individuals with (neuro-) developmental disorders. In this report, we describe three individuals with biallelic variants in POLR3GL, a gene encoding a Pol III subunit that has not been associated with disease before. Using whole exome sequencing in a monozygotic twin and an unrelated individual, we detected homozygous and compound heterozygous POLR3GL splice acceptor site variants. RNA sequencing confirmed the loss of full-length POLR3GL RNA transcripts in blood samples of the individuals. The phenotypes of the described individuals are mainly characterized by axial endosteal hyperostosis, oligodontia, short stature, and mild facial dysmorphisms. These features largely fit within the spectrum of phenotypes caused by previously described biallelic variants in POLR3A, POLR3B, POLR1C, and POLR3K. These findings further expand the spectrum of POLR3-related disorders and implicate that POLR3GL should be included in genetic testing if such disorders are suspected.

Prioritization of genes driving congenital phenotypes of patients with de novo genomic structural variants.

BACKGROUND: Genomic structural variants (SVs) can affect many genes and regulatory elements. Therefore, the molecular mechanisms driving the phenotypes of patients carrying de novo SVs are frequently unknown. METHODS: We applied a combination of systematic experimental and bioinformatic methods to improve the molecular diagnosis of 39 patients with multiple congenital abnormalities and/or intellectual disability harboring apparent de novo SVs, most with an inconclusive diagnosis after regular genetic testing. RESULTS: In 7 of these cases (18%), whole-genome sequencing analysis revealed disease-relevant complexities of the SVs missed in routine microarray-based analyses. We developed a computational tool to predict the effects on genes directly affected by SVs and on genes indirectly affected likely due to the changes in chromatin organization and impact on regulatory mechanisms. By combining these functional predictions with extensive phenotype information, candidate driver genes were identified in 16/39 (41%) patients. In 8 cases, evidence was found for the involvement of multiple candidate drivers contributing to different parts of the phenotypes. Subsequently, we applied this computational method to two cohorts containing a total of 379 patients with previously detected and classified de novo SVs and identified candidate driver genes in 189 cases (50%), including 40 cases whose SVs were previously not classified as pathogenic. Pathogenic position effects were predicted in 28% of all studied cases with balanced SVs and in 11% of the cases with copy number variants. CONCLUSIONS: These results demonstrate an integrated computational and experimental approach to predict driver genes based on analyses of WGS data with phenotype association and chromatin organization datasets. These analyses nominate new pathogenic loci and have strong potential to improve the molecular diagnosis of patients with de novo SVs.

Complementary Activity of ETV5, RBPJ, and TCF3 Drives Formative Transition from Naive Pluripotency.

The gene regulatory network (GRN) of naive mouse embryonic stem cells (ESCs) must be reconfigured to enable lineage commitment. TCF3 sanctions rewiring by suppressing components of the ESC transcription factor circuitry. However, TCF3 depletion only delays and does not prevent transition to formative pluripotency. Here, we delineate additional contributions of the ETS-family transcription factor ETV5 and the repressor RBPJ. In response to ERK signaling, ETV5 switches activity from supporting self-renewal and undergoes genome relocation linked to commissioning of enhancers activated in formative epiblast. Independent upregulation of RBPJ prevents re-expression of potent naive factors, TBX3 and NANOG, to secure exit from the naive state. Triple deletion of Etv5, Rbpj, and Tcf3 disables ESCs, such that they remain largely undifferentiated and locked in self-renewal, even in the presence of differentiation stimuli. Thus, genetic elimination of three complementary drivers of network transition stalls developmental progression, emulating environmental insulation by small-molecule inhibitors.

Mapping and phasing of structural variation in patient genomes using nanopore sequencing.

Despite improvements in genomics technology, the detection of structural variants (SVs) from short-read sequencing still poses challenges, particularly for complex variation. Here we analyse the genomes of two patients with congenital abnormalities using the MinION nanopore sequencer and a novel computational pipeline-NanoSV. We demonstrate that nanopore long reads are superior to short reads with regard to detection of de novo chromothripsis rearrangements. The long reads also enable efficient phasing of genetic variations, which we leveraged to determine the parental origin of all de novo chromothripsis breakpoints and to resolve the structure of these complex rearrangements. Additionally, genome-wide surveillance of inherited SVs reveals novel variants, missed in short-read data sets, a large proportion of which are retrotransposon insertions. We provide a first exploration of patient genome sequencing with a nanopore sequencer and demonstrate the value of long-read sequencing in mapping and phasing of SVs for both clinical and research applications.

Molecular dissection of germline chromothripsis in a developmental context using patient-derived iPS cells.

BACKGROUND: Germline chromothripsis causes complex genomic rearrangements that are likely to affect multiple genes and their regulatory contexts. The contribution of individual rearrangements and affected genes to the phenotypes of patients with complex germline genomic rearrangements is generally unknown. METHODS: To dissect the impact of germline chromothripsis in a relevant developmental context, we performed trio-based RNA expression analysis on blood cells, induced pluripotent stem cells (iPSCs), and iPSC-derived neuronal cells from a patient with de novo germline chromothripsis and both healthy parents. In addition, Hi-C and 4C-seq experiments were performed to determine the effects of the genomic rearrangements on transcription regulation of genes in the proximity of the breakpoint junctions. RESULTS: Sixty-seven genes are located within 1 Mb of the complex chromothripsis rearrangements involving 17 breakpoints on four chromosomes. We find that three of these genes (FOXP1, DPYD, and TWIST1) are both associated with developmental disorders and differentially expressed in the patient. Interestingly, the effect on TWIST1 expression was exclusively detectable in the patient's iPSC-derived neuronal cells, stressing the need for studying developmental disorders in the biologically relevant context. Chromosome conformation capture analyses show that TWIST1 lost genomic interactions with several enhancers due to the chromothripsis event, which likely led to deregulation of TWIST1 expression and contributed to the patient's craniosynostosis phenotype. CONCLUSIONS: We demonstrate that a combination of patient-derived iPSC differentiation and trio-based molecular profiling is a powerful approach to improve the interpretation of pathogenic complex genomic rearrangements. Here we have applied this approach to identify misexpression of TWIST1, FOXP1, and DPYD as key contributors to the complex congenital phenotype resulting from germline chromothripsis rearrangements.

DNA methylation restricts spontaneous multi-lineage differentiation of mesenchymal progenitor cells, but is stable during growth factor-induced terminal differentiation.

The progressive restriction of differentiation potential from pluripotent embryonic stem cells, via multipotent progenitor cells to terminally differentiated, mature somatic cells, involves step-wise changes in transcription patterns that are tightly controlled by the coordinated action of key transcription factors and changes in epigenetic modifications. While previous studies have demonstrated tissue-specific differences in DNA methylation patterns that might function in lineage restriction, it is unclear at what exact developmental stage these differences arise. Here, we have studied whether terminal, multi-lineage differentiation of C2C12 myoblasts is accompanied by lineage-specific changes in DNA methylation patterns. Using bisulfite sequencing and genome-wide methylated DNA- and chromatin immunoprecipitation-on-chip techniques we show that in these cells, in general, myogenic genes are enriched for RNA polymerase II and hypomethylated, whereas osteogenic genes show lower polymerase occupancy and are hypermethylated. Removal of DNA methylation marks by 5-azacytidine (5AC) treatment alters the myogenic lineage commitment of these cells and induces spontaneous osteogenic and adipogenic differentiation. This is accompanied by upregulation of key lineage-specific transcription factors. We subsequently analyzed genome-wide changes in DNA methylation and polymerase II occupancy during BMP2-induced osteogenesis. Our data indicate that BMP2 is able to induce the transcriptional program underlying osteogenesis without changing the methylation status of the genome. We conclude that DNA methylation primes C2C12 cells for myogenesis and prevents spontaneous osteogenesis, but still permits induction of the osteogenic transcriptional program upon BMP2 stimulation. Based on these results, we propose that cell type-specific DNA methylation patterns are established prior to terminal differentiation of adult progenitor cells. This article is part of a Special Issue entitled: 11th European Symposium on Calcium.